We propose to develop a novel method and kit for profiling the expression and activation of signal transduction proteins in breast tumors. The goal is to create new "companion" diagnostics to better select patients likely to respond to targeted therapies, including Herceptin. and novel tyrosine kinase inhibitors. 20/20 will utilize its proprietary Layered Expression Scanning (LES) technology. Transfer of antibody probes from a single tissue section to numerous bioaffinity membranes permits multiple target antigens (up to 50 if needed) to be assessed in a quantitative manner while preserving the local spatial orientation of the targets relative to the tissue section. During Phase II, we will demonstrate its tissue applications with human breast tissue sections from clinical trials in breast cancer patients treated with growth factor and Akt inhibitors. Biomarker quantification will be standardized and calibrated to clearly demonstrate a clinical benefit in comparison with subjective visual estimation. The product would have utility in both a research and clinical laboratory setting. Importantly, the system is open-ended to the addition of new prognostic markers and drug targets into the assay panel. There are several anticipated advantages of the layered membrane platform over current histopathology tools such as immunohistochemistry (IHC). These include conservation of tissue (such as for core needle biopsies) and the ability to simultaneously profile multiple signaling proteins in both normal and phosphorylated form, in a single tissue section. PUBLIC HEALTH RELEVANCE:Our options for breast cancer diagnosis and treatment will be increasingly taking advantage of information obtained from tumor profiling assays, which will indicate customized therapies for each form of the disease. The novel diagnostic techniques to be researched will allow for microscopic profiling of tiny amounts of tissue for numerous cancer-causing molecules. There will be applications in pre-clinical studies and drug trials, which will aim to invent novel treatment strategies using combinations of drugs that block `growth factor' molecules. [unreadable] [unreadable] [unreadable]